The present invention relates to an application of fatty acid CoA thioester found out as an active inhibitory substance against peroxisome proliferator-activated receptor xcex1 and xcex3 (hereinafter referred to as PPARs) to the assay of medicinal drug, and a use of fatty acid CoA thioester for medicinal drug.
The peroxisome proliferator-activated receptor (PPAR) is a transcription factor to be activated when a ligand binds to the ligand-binding domain at the side of C-termini, and one of the nuclear receptor superfamily having glucocorticoid, estrogen, thyroxine and vitamin D as ligands (Keller H. et al: Trends Endocrinol. Metab. (1993) 4, 291-296). So far, three types of isoforms of xcex1 form, xcex3 form and xcex4 form have been identified as PPARs, and the expression tissues and the functions are different respectively (Braissant O. et al: Endocrinology (1996) 137, 354-366). The PPAR xcex1 is highly expressed in the tissues with high catabolic activity of fatty acids such as liver, kidney and heart. The PPAR xcex3 is divided into PPAR xcex3 1 and PPAR xcex3 2 as two types of isoforms with the sides of different N-termini through the selection of promoters; PPAR xcex3 1 is expressed in the relatively widespread tissues and PPAR xcex3 2 is highly expressed mainly in the adipose tissue. The PPAR xcex4 is distributed in the widespread tissues.
The PPAR xcex1 binds to promoter domain of key enzymes concerning in the lipid catabolism system such as acyl-CoA synthase existing in the cytosol, acyl-CoA dehydrogenase and HMG-CoA synthase existing in the mitochondria and acyl-CoA oxidase existing in the peroxisome of liver (Schoonjans K. et al: J. Lipid Res.(1996) 37, 907-925). From the analysis of PPAR xcex1-deficient mice, it is being considered that the PPAR xcex1 plays an important role for the energy acquisition in starvation state, that is, oxidation of fatty acid and formation of ketone body in liver (Kersten S. et al: J. Clin. Invest. (1999) 103, 1489-1498).
On the other hand, it is known that the PPAR xcex3 concerns deeply in the differentiation of adipocytes (Forman BM. et al: Cell (1995) 83, 803-812). Thiazolidinedione derivatives such as troglitazone, rosiglitazone (BRL-49,653) and pioglitazone are new therapeutic drugs of type 2 diabetes with a unique function that improves the insulin resistance being one of pathogenic factors of diabetes, and, in recent years, it has been revealed that those drugs are agonists against PPAR xcex3 (Lehmann JM. et al: J. Biol. Chem. (1995) 270, 12953-12956). It is being considered that the PPAR xcex3 plays an important role for the energy storage in organisms. However, the function of PPAR xcex4 is not very understood compared with xcex1 form or xcex3 form.
As described above, for the agonists against PPAR, glitazone-classed drugs are well known. Also, it is reported that natural or endogenous-produced saturated and unsaturated fatty acids, certain kinds of eicosanoid, oxidized fatty acids, etc. are agonists against PPAR (Forman BM. et al: Proc. Natl. Acad. Sci. USA (1997) 94, 4312-4317).
On the other hand, it is the status quo that the inhibitory substance and antagonist against PPAR are little known. Only 2,4-thiazolidinedione derivatives are known as the antagonists against PPAR xcex3 (Oberfield J. L. et al; Proc. Natl. Acad. Sci. USA (1999) 96, 6102-6106).
As the use of antagonist against PPAR xcex3, application to antiobesity drug is disclosed (WO97/10813), not getting however to the discovery of antagonistic substance.
Much less, the inhibitory substance or antagonist against PPAR xcex1 is not known at all.
Up to this time, no antagonist against PPAR xcex3 and PPAR xcex1 has been discovered even in the natural or endogenous substances.
The purpose of the invention is to create a very high-novelty medicinal drug for the carbohydrate and lipid metabolism-related diseases by finding out an inhibitory substance or antagonist against PPAR xcex1 and PPAR xcex3.
When the inventors were implementing studies on the participation of PPAR in the induction of insulin resistance, they have found, to their surprise, that certain fatty acid CoA thioester forms being the metabolites of fatty acids have inhibitory function against PPAR xcex1 and PPAR xcex3, leading to the completion of the invention.
Namely, through competition binding experiments using tritium-labeled form of KRP-297 (Murakami K. et al: Diabetes (1998) 47, 1841-1847) being a dual agonist against PPAR xcex1 and PPAR xcex3, it has been found that different fatty acid CoA thioesters bind well to the ligand-binding domains of PPAR xcex1 and PPAR xcex3, thus making it clear that they are ligands of both xcex1 and xcex3 receptors.
In addition, the fatty acid CoA thioesters dose-dependently inhibited the binding activity on the conjugate formation between ligand-binding domains of PPAR xcex1 and PPAR xcex3 and steroid receptor coactivator (SRC-1). Consequently, the fatty acid CoA thioesters clarified themselves to be inhibitory substances of PPAR xcex1 and PPAR xcex3.
According to the invention, the fatty acid CoA thioester can be used for the exploration of creation of medicinal drug and the assay tools, as an inhibitory substance or antagonist against PPAR xcex1 and PPAR xcex3, which makes it useful.
Namely, the fatty acid CoA thioester in which fatty acid group is myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl or arachidonoyl can be used for the creation of medicinal drug as an inhibitory substance against PPAR xcex1, and the fatty acid CoA thioester in which fatty acid group is myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl or arachidonoyl can be used for the creation of medicinal drug as an inhibitory substance against PPAR xcex3.
Furthermore, it is also possible to use the fatty acid CoA thioester itself as a medicinal drug. Fields of medicinal drug are as follows:
It is known that, in the case of critical diabetes, mainly type 1 diabetes, the diabetic ketoacidosis can often occur as an acute complication. The diabetic ketoacidosis clinically assumes dehydration, disorder of consciousness, depressed blood pressure, tackycardia, respiratory stimulation, Kussmaul""s large respiration and acetone odor of exhalation (Keller U. et al: Diabetologia (1986) 29, 7-77). From the fact that PPAR xcex1 plays an important role for the oxidation of fatty acid and the formation of ketone body in liver, it is expected that the antagonist of PPAR xcex1 can inhibit them, hence it is useful for the therapy of diabetic ketoacidosis.
Obesity is a risk factor for diabetes, hyperlipidemia, hypertension, ischemic heart disease, etc., hence the prevention and therapy thereof are very important subjects clinically. The PPAR xcex3 plays an important role for the differentiation of adipocytes. Actually, the thiazolidinedione derivatives, PPAR xcex3 agonists, have differentiation-inducing function of adipocytes, and it is reported that they increase the number of adipocytes and the weight of adipose tissue (Piet De Vos et al: J. Clin. Invest. (1996) 98, 1004-1009). While the thiazolidinedione derivatives have usefulness as the therapeutic drugs of diabetes, they induce the differentiation of adipocytes, hence the potential for promoting the obesity is also feared. Also, it is reported that the expression level of leptin known as an antiobese factor is deprssed through the administration of thiazolidinedione derivatives (Zhang E. et al: J. Biol. Chem. (1996) 271, 9455-9459). Based on these backgrounds, the antagonist of PPAR xcex3 suppresses the differentiation of adipocytes and, at the same time, it increases the expression level of leptin, thereby the potential as an antiobesity drug is expected.